Nematodes represent a group of unsegmented round worms. They are simple in anatomy, having a simple gut and elongated fusiform shape. They are divided into numerous Families, some of which are free living while others are parasitic to plants or animals. Those which are parasitic to insects are called entomogenous or entomopathogenic nematodes.
The Order of greatest commercial interest for insect control is the Order Rhabditida, which contains several Families, many of whose members are parasitic to insects. Prominent among these Families are the Steinernematids and Heterorhabditids. A general discussion of the classification of nematodes, and the entomogenous Families thereof is found in Poinar, G. O. "The Natural History of Nematodes" (1983), Prentice-Hall, Inc., N.J.
Nematodes have a standard life cycle comprising five stages which are delineated by a molting process in which a new cuticle is formed and the old one shed. Briefly, the adults of stage 5 reproduce, and the eggs generate stage 1 larvae, which, under appropriate conditions, transit to stage 2. Normally, the stage 2 larvae simply develop to stage 3 larvae and thence to stage 4 larvae, which then complete the cycle to the adult stage. However, and of interest to the use of nematodes for insect control, when conditions are relatively unfavorable for continuing growth and reproduction, the stage 2 larvae of Steinernematid and Heterorhabditid nematodes develop instead into "stage 3 infective juveniles" or "IJs". Under these conditions, the cuticle characteristic of the second stage is retained and is called the sheath. It completely encloses the nematode. IJs are infective to insects and complete their life cycle through stage 4 and adult at the expense of the host.
Steinernematid and Heterorhabditid IJ nematodes are an effective means of insect control. They are identifiable morphologically and normally live in surface water films around soil particles. They require oxygen and moisture for survival, but do not feed; they utilize their own food reserves as an energy source. They remain infective if the sheath is removed.
One other aspect of Steinernematid and Heterorhabditid nematode biology is significant: nematodes within these families are symbiotic with species of bacteria which are primarily but not totally responsible for their entomopathogenic properties. Growth of nematodes is favored in the presence of an associated symbiont, presumably because the symbiont serves as an easily assimilated food source.
The monoxenic culture method of Bedding [(1984) Ann Appl Biol 104:117-120] has been proposed for the mass production of entomogenous nematodes. The Bedding method uses a solid phase matrix of plastic foam impregnated with homogenized animal tissue in which the nematodes are grown. The technique provides a well-aerated substrate for the growth of both the bacterial symbiont and the nematode. The Bedding process has been applied to the production of 10.sup.9 nematodes in a batch, but potential markets may require a production capacity 10,000 times this amount. A scaled up version of the Bedding process would require expensive automated equipment, would be difficult to maintain in an aseptic state, and would present difficulties in medium preparation and nematode harvesting. A liquid, monoxenic process would avoid these limitations.
Liquid, monoxenic cultures of entomogenous nematodes are usually grown in small drops of insect hemolymph. Axenic liquid cultures have been reported but are limited in efficiency and require expensive nutritional additives. For example, in the course of nutritional studies, liquid culture volumes of less than 100 ml were used by Stoll, N. (1961) J Helminthol, Lister Suppl, pp. 169-174; Jackson, G. J. (1973) Exp Parasitol 34:111-114; Hansen, E., et al (1967), 42nd Ann Meeting Am Soc Parasitol; Lower, W. M. R., et al (1970) Nematologia 16:563-566;,and Beucher, E. J., et al (1970) Nematologia 16:403-409. Nevertheless, these reports demonstrate that the biological and physiological requirements of the nematodes can be met in liquid suspension. Some means for aeration of these cultures is required and may be supplied by either diffusion into thin liquid layers, shaking, or bubbling.
One of the greater difficulties in developing a liquid, monoxenic culture is in providing sufficient aeration for both the bacteria and the nematodes without exposing the nematodes to excessive shear forces. One technique has been reported for the growth of C. elegans, a free-living nematode, in liquid, monoxenic culture. In this technique, bacteria are suspended with nematodes in a non-nutrient salt solution. Under these conditions, the bacteria have little free nutrient to metabolize and therefore have a low oxygen demand. During studies leading to the present invention, it was found that this same technique could be used for the culture of N. carpocapsae as long as a sterol additive was provided. The bacterial requirement was found to be so high, however, that commercial exploitation of the technique was economically precluded.
PCT Patent Application No. 86/01074, published Feb. 27, 1986 (hereinafter referred to as "Pace et al"), described a process for large-scale culture of nematodes which addresses some of the problems associated with aeration requirements. This procedure determined the stirring rate in a stirred reactor at which adult nematodes are disrupted. The stirring rate was then set at a level below this determined rate and maintained throughout the culture period.
Pace et al also describe the use of a conventional liquid culture medium composed of ox kidney homogenate and yeast extract. Other media for nematode culture have included soy peptone-yeast extract-dextrose (Buecher et al (1971) J Nematol 3:199-200 for use in axenic liquid culture); peptone-glucose and bacteria (Dutky, S. R., et al (1967) J Nematol 13:140 for monoxenic culture on agar); and nutrient broth-yeast extract-soy flour-corn oil (Wouts, W. M., et al (1981) J Nematol 13:467-469, for monoxenic solid support or foam). Each of these media contain one or more ingredients that is expensive or difficult to prepare.
The monoxenic liquid culture method of Pace et al gave a reproductive rate in the first 10 days of 20 (an increase from 2,000 to 40,000 nematodes per milliliter) and reported the formation of infective juveniles in liquid culture after 20 days. Other investigators using axenic systems have reported similar reproductive rates, but did not report observing significant levels of infectious juveniles.
It has been discovered that a critical component in a liquid, nutrient culture medium supportive of monoxenic nematode culture but controlled for oxygen demand, is the requirement of an emulsifier, such as egg yolk, which serves to facilitate the utilization of added oils or fats. To date, there have not been any reports using an emulsifier to benefit liquid culture of entomogenous nematodes; however, egg yolk has been used as a replacement for animal serum in the culture of mammalian parasites and has met with both success and failure.
A. Roder [(1982) Naturwissenschaften 69:92-93] used egg yolk to replace fetal calf serum in the culture of insect cells. M. S. Schnier and B. Fried [(1981) Int J Parasitol 10:391-395] used egg yolk in NCTC 135 in the culture of Ablosoma suwaense, a parasitic trematode (a worm in the fluke phylum). In addition, egg yolk has frequently been used in bacterial cell cultures especially with Staphylococcus. P. F. Busch et al [(1973) J Parasitol 59:319-322] found that egg yolk did not benefit Cotylurus (a trematode) culture and D. W. W. Kannangara [(1974) Int J Parasitol 4:675-6] found that egg yolk did not benefit Paragonimus culture. None of these cases involved a role for egg yolk in emulsifying an oil component.
It is, therefore, an object of the present invention to improve.. the production efficiency of entomogenous nematodes in liquid culture.
It is a further object of the invention to provide an improved liquid culture medium support of monoxenic nematode culture but controlled for oxygen demand. This nutrient medium is easily prepared and is relatively inexpensive to produce as compared to presently available media.
Yet a further object of the invention provides a method to enhance bacterial and nematode development wherein the agitation rate is varied depending on the changing oxygen transfer requirements associated with each nematode developmental stage.